Introduction:
This month's MOTM is an inorganic cubane complex known as the manganese-calcium oxide
cluster, commonly referred to as the "Oxygen Evolving Complex" or
OEC (also referred to as a water oxidase). The OEC is found on the oxidizing side of Photosystem II (PSII), and is
located within organelles known as chloroplasts in all plants and algae.
The OEC is also found in one group of bacteria, the Cyanobacteria (formally called the "blue-green algae", a name
now discounted since the Cyanobacteria are now known not to be prokaryotic bacteria). It is believed that the Cyanobacteria are the endosymbiotic ancestors of modern
day chloroplasts. (1) In the case of higher plants and algae, the
OEC is found integrally embedded within the thylakoid membranes,
located throughout a plant cell's chloroplasts. Cyanobacteria
have no internal organelles, and therefore no chloroplasts, and the OEC is found within thylakoid membranes
distributed throughout the cytoplasm. The
Cyanobacteria are quite small, and are about the same size as the internal chloroplasts of the eukaryotic plants and algae. This is
one branch of evidence which supports the theory that chloroplasts are ancient Cyanobacteria (many other facts exists which also support this
theory (circular DNA, bacterial ribosomes, et al.) (2)
To review the components of PSII, click on the image at left to explode the details of the PSII reaction center.

There are two photosystems, PSII and PSI, which communicate with one another
via an electron transport chain known as the "Z scheme" of
photosynthesis. Electrons are initially extracted from water, one at a time (1 photo per electron) at the site of the OEC, and finally released to a soluble ferredoxin
complex after passing through PSI (where it is further re-energized by a second photon). Ferredoxin then reduces Fd-NADP
oxidoreductase, which reduces oxidized NADP, which then reduces 1,3-bisphosphoglycerate. This reduction step regenerates
Ribulose-5-phosphate followed by incorporation of CO2 by Ribulose-bisphosphate carboxylase (Rubisco) -
the most abundant protein on the planet). The reduction of CO2(carbon fixation) is known as the "dark reaction" or Calvin Cycle (sugar production).

In algae and plants the thylakoid membranes are found partially stacked (grana) where PSII occurs in higher concentration than PSI (the stacking of the thylakoids may be related to an increase in the photon transfer efficiency by the
surrounding light harvesting chlorophyll complexes - in the pop up of the light harvesting complexes, notice the concentric arrangement of protein, chlorophyll (and carotenoids), centered around the PSII reaction center.

Recently the three dimensional atomic structure of the manganese-calcium oxide complex was determined by X-Ray diffraction.
(3) The manganese cluster is essentially an inorganic cubane (cubical) structure found in inorganic crystals
made of ranciéite or hollandite
[Mn4CaO9*3H2O].
These minerals are believed to have been assimilated during the early Archaean period by the Cyanobacteria approximately 3,200 - 2,800 million years
ago (or 3.2 - 2.8 Ga=giga years ago). The assimilation of the manganese-calcium oxide complex by the Cyanobacteria is one of several examples
of metallo-catalysts incorporated by living systems. Two other inorganic metallo complexes assimilated by early prokaryotes (and are now
ubiquitous redox centers in living systems) include the cubic iron-sulfur centers [Fe4-S4]
[image]
(originating as greigite (Fe4S4][SFeS]2)(4), such as ferredoxin, and the nickel-iron centers
[NiFe][image], such as Hydrogenases.

The successful assimilation of the OEC by early Cyanobacteria resulted in an explosion of this group which became the predominant life form for 2 billion years. Unlike other photosynthetic
bacteria (discussed briefly below) the Cyanobacteria are "oxygenic photosynthesizers" capable of oxidizing water as a substitute for
other electron donors such as H2S and organics (acetate, succinate, e.g.), producing molecular oxygen as a by-product. Oxygen irreversibly changed the course of life on earth by
releasing O2 in great quantities (changing, for example, the composition of the atmosphere as well as earth's geology). During this time the
Cyanobacteria successfully dispersed globally and diversified through evolutionary speciation. (5) Finally,
member(s) of the Cyanobacteria symbiotically merged with eukaryotes at about 2.0 Ga to become the chloroplast of all algae and higher plant
groups on the earth today. The arrival of the OEC of PSII resulted in the most profound impact on the history of
evolution. Indeed, the OEC is single-handedly responsible the rise of all plants and animals on the earth today.

Early Earth:
It is estimated that earth's oceans, continents and atmosphere required approximately 600 million years to stabilize (4.6 - 4.0 Ga) to
the point where liquid water accumulation and continental crust accretion were possible. (3)
Some reports put this time as early as 4.4 Ga. (4).
Fig 1. is an illustration of the earth relatively soon after it coalesced. Surface temperatures would have been very high, as well as meteorite impact frequency (although in time the meteor impact
frequency would decrease to about one impact per century by 4.0 Ga). (5) (Fig 2). This early stage
of the earth's history is known as the Hadean Eon, and it was towards the end of this eon that the continents were formed and
the oceans accumulated (approximately 4.0 Ga).

Early chaotic surface events, though becoming less frequent with time, included meteor bombardment, unstable temperatures, strong lunar forces, tides and storms, risings oceans, reduced sunlight intensity, short day length, UV,
X-Ray and other high energy irradiation, volcanic and geologic upheavals and an ever changing atmospheric chemistry (6).

Earth's atmosphere at the beginning of the Hadean period (4.6 - 4.2 Ga) was strictly reducing (lacked
oxygen) (7), having measurable levels of H2, NH3, CH4 and
H 2S. Outgassing by volcanic and thermal vent activity released reduced metals, especially Iron, as well as Phosphorous,
Sulfur, H2, H2S, CH4 and CO2
resulting in an acidic ocean. (8)
Due to occasional extreme temperature conditions, exceeding 800oC (9),
as well as active solar flaring (10) and other high-energy events, it is believed that CH4
and NH 3 would have been heat destroyed. So long as molecular oxygen was absent, the earth's atmosphere remained in a reduced state. (11)
By around 4.2 Ga, the earth's atmosphere was becoming relatively stable and consisted primarily of volcanic gas. At this time the atmosphere was primarily water
vapor (94%), CO (4%), N 2(4%), CO, and SO 2 (12).
Fig 2. represents an image of the earth at about 4.0 Ga, when oceans were deposited, continents formed, and periodic meteorite strikes were
diminishing. Fig 3 is an artist's rendition of the earth at about 3.8 Ga (note that the moon is much closer) which corresponds to the time that Life first
appeared on the earth. (13)
Hydrothermal vents and volcanic outgassing created a large reservoir
of oceanic Fe(II) and FeS. Oxidation of metals by UV radiation in
oceanic surface waters may have
resulted in an accumulation of Fe and Mn oxide precipitates, which
subsequently settled out. (14)
These early oxidized metals, such as Fe(III), may have served as life's
first electron acceptors.

First Life: The first organisms were extremophilic chemotropic prokaryotes (that is, tolerated
extreme environmental conditions such as high heat, high salt concentration, high and low pH, etc). The extremophiles are
now known to have been early members of the domain Archaebacteria, although it is argued that simultaneous
evolution of the eubacteria as well as the eukaryotes paralleled these early extremophiles (15). Many species
of the archaebacteria survive today (such as the acetogens,
methanogens,
thermophiles and
halophiles). These organisms, like true bacteria, lack a nucleus, as well as organized internal membrane structures, vacuoles
and internal organelles .

The early archaebacteria did however have an amazing array of biochemical pathways to derive energy, acquire carbon, nitrogen, phosphorous, sulfur, hydrogen,
bicarbonate and various organics as
well as the requisite minerals needed for growth. In addition they had RNA and DNA, giving them the ability to store and pass on genetic information necessary to build enzymes to carry out the multitude of
necessary biochemical processes for life. In addition, the early prokaryotes had the capacity (as extant members of the Archaebacteria have today) to "detect" and "respond"
to changes in their environment, such as redox potentials, nutrient levels, physical events, light and temperature variances, et al. The ability to respond to changes in their immediate environments allowed them to adjust,
tolerate, thrive, reproduce and evolve. The various hypothetical pathways
which led to earth's first life forms from proto-life is an area of active and intense research. It is believed that the first organisms were
acetogens or closely related methanogens [see Timeline below].

The oldest fossils of life on earth are found in microfossils (3.8 Ga) and stromatolites (3.5 Ga). As shown in (Fig 5, 6 and 7) Stromatolites appeared in the early shallow waters
of the earth approximately 3.8 Ga. The artist's rendition in Fig. 5 (below, left) of the earth at that time accurately depicts the volcanic outgassing and undersea hydrothermal
vent activity which would have been prevalent near the end of the Hadean eon. The earliest stromatolites were probably pre-Photosynthetic eubacteria such as
Chloroflex
and Chlorobium, both photosynthetic archaean anaerobic thermophiles which preceded the Cyanobacteria.

The assimilation of the OEC by the cyanobacteria laid the ground work for the Cambrian explosion, and for the evolution of the animal and plant kingdoms.
Cyanobacteria, by assimilating the OEC and releasing molecular oxygen, changed the geological landscape of the earth, as well as its atmosphere and
oceans. All other organism would respond in abeyance, either taking up oxygenic respiratory
or related metabolism, or recess into niches
devoid of oxygen's diffusional persistence.

Recent genomic mapping and macromolecular resolution (especially by X-Ray diffraction) as well as a host of other approaches (kinetic studies, et al.) has led to an
explosion of research on the evolution of life. The recent results of these efforts,
brought about by the computer and advanced technological research capabilities, have been staggering. Previous concepts of taxonomy, based primarily on
morphology and physiology, have been supplanted by the work of Woese and others
who revolutionized the concept of DNA as molecular fossils
(16). Woese and his group are
responsible for the discovery of the domain Archaea, and in the process laid the ground work for
future research in this area (Woese's work gives
new meaning to the saying "..when one door is closed, many more are open.").

2. Bacterial Photosynthesis (3.8 - 2.7 Ga)

Introduction to the
Photosynthetic bacteria: Thirty-six bacterial lineages (Eubacteria) have been identified, of which only five are capable of using chlorophyll-based energy conversion
to create a Protonmotive Force (PMF) to drive ATP synthesis and reduce
CO2 to sugars. Of the five bacterial groups capable of photoautotrophic
photosynthetic growth, four, (the exception being the Cyanobacteria), perform photosynthesis under anaerobic conditions and do not oxidized water to molecular oxygen
via the OEC (anoxygenic photosynthesis). Indeed, the
Cyanobacteria are the only group of bacteria which have assimilated the
OEC necessary for splitting
water. Members of the five photosynthetic bacterial families are shown below (these are just example species from each group):

One of the most fascinating areas of biochemistry today is research designed to determine the various evolutionary relationships
among these lineages and their evolutionary origins (as well as all of the Eubacteria). Recent findings have revealed that these groups are highly
heterophyletic. Scientists now believe that the transfer of genetic information between divergent organisms has been prolific, especially early in evolution, thereby complicating further the genomic mapping of their
ancestry (genes may be transferred between divergent organisms though such processes such as
conjugation. (17) These events have literally turned the "tree of
life" into a three dimensional "web of life." With species dying out in the interim, and
evolution spanning more then 3,000 million years, biochemical
researchers certainly have their work cut out in the foreseeable future.

All known microorganisms use two functional principles (both mutually exclusive and represent two independent evolutionary developments) for
the conversion of light into chemical energy. Chlorophyll-based systems are widespread among members of the domain bacteria and consist of a
light-harvesting antenna and reaction centers. In the latter, excitation energy is converted into a redox gradient across the membrane

Bacterial reaction center shown above, left. Note
the Bacteriachlorophyll reaction center "dimer", a special
pair of chlorophyll molecules designed to absorb incoming photons from
surrounding light harvesting chlorophyll complexes, shown on the
right. Blue chlorophylls shown in the reaction center at left are
pheophytins, bacteriachlorophyll minus the Mg atom inside the
chlorophyll heme. Red molecules are quinones (the extended tails
of the quinones allows for lateral motion and membrane
solubility). The electron path begins with
the oxidation of the dimer pair of chlorophylls (the reaction center
proper) followed by electron transfer through the pheophytins and onto
the quinones. The reaction center dimer is then reduced by an
external electron sources such as Cytochrome.

Phycobilisomes: Phycobilisomes
are light harvesting pigments found throughout the Cyanobacteria (and in
red algae). All other photosynthetic bacteria use the light
harvesting complex as described above. Phycobilisomes, which
specialize in absorbing deep penetrating green light (more than 1 meter
deep, e.g., in marine environments), are capable of re-emitting light in
regions which the other photosynthetic pigments can absorb.
This would be a very advantageous adaptation in marine environments in
which protection from UV radiation might be needed. There is a
central core of allophycocyanin (AP) which sits above the photosynthetic
reaction center. There are phycocyanin and phycoerythrin subunits which
radiate out from this center like thin tubes. The fluorescent pigments
which are present in the phycobilisome, such as phycocyanobilin (PC) and
phycoerythrobilin (PE) re-emit the green light in regions.

Chlorosomes: Model of the photosynthetic apparatus in Chlorobium
tepidum showing "chlorosome" body compiled by Dr. Donald Bryant and associates at Penn State
University (Chlorosomes found in the green sulfur bacteria, family
Chlorobiaceae). Light and excitation energy transfer is shown in red,
electron transfer is shown in blue. Proteins and chlorosome rod elements
are shown in color and some cofactors are indicated as white symbols.
Diamonds represent individual BChl c molecules in the chlorosomal rod
elements, BChl a molecules in CsmA, FMO protein, and reaction center
(the double diamond represents the P840 special pair), and the primary
acceptor Chl aPD in the reaction center; cubes represent
[4Fe-4S] clusters in the reaction center and soluble ferredoxin;
rhomboids represent [2Fe-2S] clusters in CsmI, CsmJ, CsmX, and PetC;
squares represent heme groups in the reaction center, PetB, and soluble
cytochrome c; double hexagons represent menaquinone molecules in the
reaction center and cytoplasmic membrane; triple hexagons represent a
FAD cofactor.

The evolution of two Photosystems:
The evolution of the oxygenic photosynthetic reaction center is
currently under intense research scrutiny. There are two
photosystems, known as Reaction Center Type I (RC1 or PSI)
and Reaction Center Type II (RC II or PSII). The OEC became
associated with RC II, but we find that both Photosystems are used by
prokaryotes with slightly differing functions and evolutionary
adaptations. Cyanobacteria use the oxygenic PSII and PSI electron
transport system as does algae and plants. Anoxygenic bacteria use
either RC I, or a prototype of RC II, depending on species and environment.
At least one organism, Oscillatoria, has both.

To understand the various distribution and functions of
the two reaction centers, and how they might be related evolutionarily,
John F. Allen (Dept of Biology, University of London) recently published
a model to explain the appearance of the two reaction centers throughout
the prokaryotes and higher plants and algae. It is now generally accepted that PSII
evolved from a primitive PSI, the
two reaction centers are in fact homologous in both function and
structure (determined from kinetic, X-Ray and genetic studies). Dr.
Allen calls his model a "redox switch hypothesis" in which the
redox levels in the environment (amount of available reductants, etc)
trigger a bacteria to switch from one reaction center to another.
The associated light harvesting chlorophyll proteins, being integral
within the bilipid membrane, are capable of serving as light harvesting
complexes for both photosystems. Dr. Allen's model is presented
below.

The original RC I is shown in the lower left, labeled as
proto-RC. This reaction center operates with external reductants,
such as hydrogen sulfide, and is capable of supplying electrons to a
quinone pool, which can then recycle the energized electrons back to the
reaction center while moving protons across the membrane into the
periplasmic space thereby creating a protomotive force for the synthesis
of ATP. In addition, the RC I provided electrons to soluble
electron acceptors (ferredoxin) for use in organic synthesis.

Over time and changing conditions, a second independent
photosynthetic reaction center emerged, under a genetic control scheme
which would favor one over the other. Allen argues that in this
early stage the two reaction centers would have been cooperative, their
expression genetically controlled by changing environmental conditions
(alternatively, there may have been initial competition). The rise
of the RC II enabled the bacteria, under conditions in which a
reductant, such as H2S,
was readily available to support photon-driven proton pumping through
the quinone cycle into the periplasmic space for ATP synthesis.
Single reaction center anaerobic phototropohs were either
photolithotrophic (RC 1) or photoorganotrophic (RC II). Dr. Allen
argues that metabolic flexibility may have selected those bacterial
capable of providing one or both of the two types of photosystems,
depending on environmental conditions.

Over time bacteria appeared (such as in the present day Oscillatoria) in which both photosystems are
present, and are synthesized and distributed through the membrane
depending on environmental conditions encountered (Allen proposes this
is carried out under redox control of gene expression). Oscillatoria
limnetica has both RC I and RC II, and under conditions of low
H2S, switches to RC II and performs oxygenic photosynthesis
(Oscillatoria is a member of the Cyanobacteria. Note that there are other
conditions, such as the heterocyst of Nostoc, in which RC II is shut
off, as oxygen will interfere and shut down nitrogen fixation. See
also C4 cell et al.).

As the image above shows, bacteria with RC II only are
known (anoxygenic), as well as bacteria with RC I only (also anoxygenic)
Dr. Allen proposes that at some point in time the genetic "redox
switch" was not longer needed and the two photosystem began to
specialize and work in cooperation. Genetic regulatory control can
still take place by either lowering or increasing either the reaction
centers themselves or distributing their associated light harvesting
complexes between the two.

4. The
molecular structure of the manganese-calcium oxide water splitting
complex

[This section will be complete
June 2d, in the am]

Resolution:
The recent 3.5 Angstrom resolution of the OEC has opened a black box
that has been closed for many years. Earlier kinetic and
spectroscopic data are falling in line with the mechanism of this
complex but the race is on to determine exactly how the oxygen evolving
complex works. We now know there are 4 manganese atoms (their
respective redox states and behavior has not been determined with
certainty), a calcium atom, now believed to be an essential component of
the water oxidizing site, and a chloride (Cl-) co-factor. Chloride
does not appear to be essential but is believed to be a charge
stabilizing co-factor. An excellent animation of the Photosystem
II complex, including the light harvesting chlorophylls, the reaction
center and the OEC can be downloaded at
Dr. Johannes Messinger's site (36 megs, Windows, RealTime) and is a beautiful rendition of PSII
(highly recommended).

Dr. Messinger suggests that once the mechanism of water
oxidation has been determined, it may be used to engineer an energy
source by combining the oxidation of water with a hydrogenase (future
solar hydrogen and oxygen production).

The mechanism of water oxidation has eluded scientists
for decades. In the last 50 years much work has been carried out
on the kinetics of PSII (OEC). In addition electrophoresis and related techniques carried out
over the last 30 years had identified most of the polypeptides
associated with PSII. New X-Ray diffraction studies have
identified the stereochemistry and atomic structure of the OEC and surrounding complexes.
It appears now that the
race is on to determine the mechanism of water splitting (one of
biology's greatest secrets). Once the mechanism of water oxidation
is elucidated, it will become the greatest milestone of biochemistry
since the discovery of DNA's structure in 1954.

Photosystem
II consists of an array of proteins, held together by numerous salt
links, hydrogen bonds, water molecules et al. and can become especially
labile under stressful conditions such as high temperatures and low
pH. The complex is also sensitive to photoinactivation if electron
flow throw the "Z" scheme is not delicately balanced (ATP
phosphorylation of the surrounding light harvesting chlorophyll proteins
is part of this regulatory scheme (x)).

From the image at left, PSII is diagrammatically
depicted as a dimer, as it is found embedded in the thylakoid membranes
(see Dr. Messinger's video, which clearly shows the central axis of this
complex). The PSII reaction center complex is surrounded by
additional light harvesting chlorophylls, shown here as LHCII (only 2
are shown, these would completely surround the complex in a natural
state.

The
various proteins that surround the PSII complex are shown in the diagram
at left. Then include the principle Light Harvesting Complexes
(LHCI), D1, and D2, as well as associated peptides including the 33
kdal, 23 kdal and 17 kdal. These periphery proteins support the
reaction center and associated cytochromes and other pigments.

The general reaction is also outline (detailed below) in
which the reaction center chlorophyll dimer, P680, is initially oxidized
by an incoming photon, the electron being passed to the nearby
pheophytin and then onto the quinone pool. Manganese, bound to
water, calcium, chloride and oxygen (in manganese oxo bridges) then
rapidly reduces Yz (a local Tyrosine) which itself became oxidized when
it reduced the oxidized P680+. The basic process of water
oxidation's mechanism was worked out 35 years ago by Joliot (X)
and Kok (Y). Each PSII reaction center within
the membrane works independently of one another and cycles through a set
of 5 oxidation states, known as the "S" states (So
-> S4).
Our current understanding of the mechanism of water oxidation proceeds
through these four states known as the "Kok Scheme" (for more
details on the structure of PSII proper see the references and links
provided below.

The X-Ray data and model
of the OEC are shown below.

The original X-Ray at a resolution of 3.5 Angstroms, was
reported by Ferreira et al in 2004. The image to the left, above,
was taken from an article published by James P. McEvoy, Yale University
(X). The image was colored coded and the
middle image drawn directly from that. The model on the right was
built using this X-Ray information.

The salient features of the complex (not all natural
ligands may be in place, and McEvoy added an additional water ligand to
complete the complex) include the manganese-calcium oxo bridging of the
complex. The manganese atoms are labelled 1, 2, 3 and 4, with manganese
number 4 protruding outside the main cubical structure below it, which
includes manganese oxo binding, between the Mn 1, Mn 2 and Mn 3.
Calcium is placed as one of the cuboidal corners (shown in blue on the
left). Chloride is believed to reside on the outside of this
cubane complex, and in our model on the right, appears to sit nicely
(electrostatically) amongst the water molecules and calcium that
surround it.

Yz, the primary electron acceptor of the OEC, sits to
the left, approximately 3.4 Angstroms away from the chloride. Note
the numerous ligands supporting the complex, which includes several
aspartates (contributed from the nearby D1 protein) as well as a
histidine residue (near the bottom), also from D1. The substrate
water molecules, labeled W1 and W2 in our model (right) is, at this
time, an arbitrary assignment since the exact details of the mechanism
is not yet known (we chose these water molecules after the model was
built because of their proximity to Mn 4, and to illustrate a potential
source of oxygen in our mechanistic scheme below. [when this article is
complete tomorrow am (6-2) these images will be clickable to yield a
closer view]. A proposed water channel may be directed out from
aspartate 170 (D1) since it is believed that Tyrosine and histidine
(left of the manganese cube) reside in a hydrophobic environment.

[At press time this page was not complete, and will be
completed later today - final page should be up first thing on the
morning of June 2d, tomorrow (please return then for a closer look).]

Water oxidizing Mechanism here:

[Mechanistic proposal to be on line on morning of June
2d [page not completely finished in time for Press]